Isolated, truncated nucleic acid molecules which code for gage tumor rejection antigen, the tumor rejection antigen, and uses thereof

ABSTRACT

A new family of tumor rejection antigen precursors, and the nucleic acid molecules which code for them, are disclosed. These tumor rejection antigen precursors are referred to as GAGE tumor rejection antigen precursors, and the nucleic acid molecules which code for them are referred to as GAGE coding molecules. Various diagnostic and therapeutic uses of the coding sequences and the tumor rejection antigens, and their precursor molecules are described. Tumor rejection antigens are also shown.

RELATED APPLICATION

This application is a Divisional of Ser. No. 08/370,648 filed Jan. 10,1995 now U.S. Pat. No. 5,648,226 which in turn is a continuation-in-partof Ser. No. 08/250,162 filed May, 27, 1994, now U.S. Pat. No. 5,610,013which is a continuation-in-part of Ser. No. 08/096,039 filed Jul. 22,1993 (abandoned).

FIELD OF THE INVENTION

This invention relates to a nucleic acid molecule which codes for atumor rejection antigen precursor. More particularly, the inventionconcerns genes, whose tumor rejection antigen precursor is processed,inter alia, into at least one tumor rejection antigen that is presentedby HLA-Cw6 molecules. The genes in question do not appear to be relatedto other known tumor rejection antigen precursor coding sequences. Theinvention also relates to peptides presented by the HLA-Cw6 molecules,and uses thereof.

BACKGROUND AND PRIOR ART

The process by which the mammalian immune system recognizes and reactsto foreign or alien materials is a complex one. An important facet ofthe system is the T lymphocyte, or "T cell" response. This responserequires that T cells recognize and interact with complexes of cellsurface molecules, referred to as human leukocyte antigens ("HLA"), ormajor histocompatibility complexes ("MHCs"), and peptides. The peptidesare derived from larger molecules which are processed by the cells whichalso present the HLA/MHC molecule. See in this regard Male et al.,Advanced Immunology (J.P. Lipincott Company, 1987), especially chapters6-10. The interaction of T cells and HLA/peptide complexes isrestricted, requiring a T cell specific for a particular combination ofan HLA molecule and a peptide. If a specific T cell is not present,there is no T cell response even if its partner complex is present.Similarly, there is no response if the specific complex is absent, butthe T cell is present. This mechanism is involved in the immune system'sresponse to foreign materials, in autoimmune pathologies, and inresponses to cellular abnormalities. Much work has focused on themechanisms by which proteins are processed into the HLA bindingpeptides. See, in this regard, Barinaga, Science 257: 880 (1992);Fremont et al., Science 257: 919 (1992); Matsumura et al., Science 257:927 (1992); Latron et al., Science 257: 964 (1992). Also see Engelhard,Ann. Rev. Immunol. 12: 181-207 (1994).

The mechanism by which T cells recognize cellular abnormalities has alsobeen implicated in cancer. For example, in PCT applicationPCT/US92/04354, filed May 22, 1992, published on Nov. 26, 1992, andincorporated by reference, a family of genes is disclosed, which areprocessed into peptides which, in turn, are expressed on cell surfaces,which can lead to lysis of the tumor cells by specific CTLs cytolytic Tlymphocytes, or "CTLs" hereafter. The genes are said to code for "tumorrejection antigen precursors" or "TRAP" molecules, and the peptidesderived therefrom are referred to as "tumor rejection antigens" or"TRAs". See Traversari et al., Immunogenetics 35: 145 (1992); van derBruggen et al., Science 254: 1643 (1991), for further information onthis family of genes. Also, see U.S. patent application Ser. No.807,043, filed Dec. 12, 1991, now U.S. Pat. No. 5,342,774.

In U.S. patent application Ser. No. 938,334 now U.S. Pat. No. 5,405,940,the disclosure of which is incorporated by reference, it is explainedthat the MAGE-1 gene codes for a tumor rejection antigen precursor whichis processed to nonapeptides which are presented by the HLA-A1 molecule.The reference teaches that given the known specificity of particularpeptides for particular HLA molecules, one should expect a particularpeptide to bind to one HLA molecule, but not to others. This isimportant, because different individuals possess different HLAphenotypes. As a result, while identification of a particular peptide asbeing a partner for a specific HLA molecule has diagnostic andtherapeutic ramifications, these are only relevant for individuals withthat particular HLA phenotype. There is a need for further work in thearea, because cellular abnormalities are not restricted to oneparticular HLA phenotype, and targeted therapy requires some knowledgeof the phenotype of the abnormal cells at issue.

In U.S. patent application Ser. No. 008,446, filed Jan. 22, 1993 andincorporated by reference, the fact that the MAGE-1 expression productis processed to a second TRA is disclosed. This second TRA is presentedby HLA-C clone 10 molecules. The disclosure shows that a given TRAP canyield a plurality of TRAs.

U.S. patent application Ser. No. 994,928, filed Dec. 22, 1992, andincorporated by reference herein teaches that tyrosinase, a moleculewhich is produced by some normal cells (e.g., melanocytes), is processedin tumor cells to yield peptides presented by HLA-A2 molecules.

In U.S. patent application Ser. No. 08/032,978, filed Mar. 18, 1993, andincorporated by reference in its entirety, a second TRA, not derivedfrom tyrosinase is taught to be presented by HLA-A2 molecules. The TRAis derived from a TRAP, but is coded for by a non-MAGE gene. Thisdisclosure shows that a particular HLA molecule may present TRAs derivedfrom different sources.

In U.S. patent application Ser. No. 08/079,110, filed Jun. 17, 1993 andincorporated by reference herein, an unrelated tumor rejection antigenprecursor, the so-called "BAGE" precursor is described. The BAGEprecursor is not related to the MAGE family.

The work which is presented by the papers, patent, and patentapplications cited supra deals, in large part, with the MAGE family ofgenes, and the unrelated BAGE gene. It has now been found, however, thatadditional tumor rejection antigen precursors are expressed by cells.These tumor rejection antigen precursors are referred to as "GAGE" tumorrejection antigen precursors. They do not show homology to either theMAGE family of genes or the BAGE gene. Thus the present inventionrelates to genes encoding such TRAPs, the tumor rejection antigenprecursors themselves as well as applications of both.

The invention is elaborated upon further in the disclosure whichfollows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, B, C, and D set forth lysis studies using CTL clone 76/6.

FIG. 2 shows tumor necrosis factor ("TNF") release assays obtained withvarious transfectants and controls.

FIG. 3 compares lysis induced by cytolytic T lymphocytes of clone CTL76/6. Peptides of varying length were tested, including SEQ ID NOS: 4-8and 12.

FIGS. 4A and B present an alignment of the cDNAs of the six GAGE genes(SEQ ID NOS: 1 and 14-18) discussed herein. In the figure, identicalregions are surrounded by boxes. Translation initiation sites and stopcodons are also indicated. Primers, used in polymerase chain reaction asdescribed in the examples, are indicated by arrows.

FIG. 5 sets forth the alignment of deduced amino acid sequences for themembers of the GAGE family (encoded by SEQ ID NOS:1 and 14-18).Identical regions are shown by boxes, and the antigenic peptide of SEQID NO: 4, is shown.

FIG. 6 shows the results obtained when each of the GAGE cDNAs wastransfected into COS cells, together with HLA-Cw6 cDNA. Twenty-fourhours later, samples of CTL 76/6 were added, and TNF release wasmeasured after twenty-four hours.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Example 1

A melanoma cell line, MZ2-MEL was established from melanoma cells takenfrom patient MZ2, using standard methodologies. This cell line isdescribed, e.g., in PCT Application PCT/US92/04354, filed May 22, 1992,published Nov. 26, 1992, and incorporated by reference in its entirety.Once the cell line was established, a sample thereof was irradiated, soas to render it non-proliferative. These irradiated cells were then usedto isolate cytolytic T cell clones ("CTLs") specific thereto.

A sample of peripheral blood mononuclear cells ("PBMCs") was taken frompatient MZ2, and contacted to the irradiated melanoma cells. The mixturewas observed for lysis of the melanoma cells, which indicated that CTLsspecific for a complex of peptide and HLA molecule presented by themelanoma cells were present in the sample.

The lysis assay employed was a chromium release assay following Herin etal., Int. J. Cancer 39:390-396 (1987), the disclosure of which isincorporated by reference. The assay, however, is described herein. Thetarget melanoma cells were grown in vitro, and then resuspended at 10⁷cells/ml in DMEM, supplemented with 10 mM HEPES and 30% FCS, andincubated for 45 minutes at 37° C. with 200 μCi/ml of Na(⁵¹ Cr)O₄.Labelled cells were washed three times with DMEM, supplemented with 10mM Hepes. These were then resuspended in DMEM supplemented with 10 mMHepes and 10% FCS, after which 100 ul aliquots containing 10³ cells,were distributed into 96 well microplates. Samples of PBLs were added in100 ul of the same medium, and assays were carried out in duplicate.Plates were centrifuged for 4 minutes at 100 g, and incubated for fourhours at 37° C. in a 8% CO₂ atmosphere.

Plates were centrifuged again, and 100 ul aliquots of supernatant werecollected and counted. Percentage of ⁵ Cr release was calculated asfollows: ##EQU1## where ER is observed, experimental ⁵¹ Cr release, SRis spontaneous release measured by incubating 10³ labeled cells in 200ul of medium alone, and MR is maximum release, obtained by adding 100 ul0.3% Triton X-100 to target cells.

Those mononuclear blood samples which showed high CTL activity wereexpanded and cloned via limiting dilution, and were screened again,using the same methodology. The CTL clone MZ2-CTL 76/6 was thusisolated. The clone is referred to as "76/6" hereafter.

The same method was used to test target K562 cells, as well as themelanoma cell line. FIG. 1 shows that this CTL clone recognizes andlyses the melanoma cell line, i.e. MZ2-MEL but not K562. The clone wasthen tested against other melanoma cell lines and autologousEBV-transformed B cells in the same manner described supra. FIG. 1 showsthat autologous B cells, transformed by Epstein Barr Virus ("EBV") werenot lysed, and that while MZ2-MEL 3.0 was lysed by CTL clone 76/6, thecell line MZ2-MEL.4F⁻, a variant which does not express antigen F, wasnot. Hence, the clone appears to be specific for this antigen.

The results presented supra are inconclusive as to which HLA moleculepresents the TRA. The lysed cell line, i.e., MZ2-MEL, is known toexpress HLA-A1, HLA-A29, HLA-B37, HLA-B44, HLA-Cw6, and HLA-C clone 10.In experiments not reported here but which followed the protocol of thisexample, a subline of MZ2-MEL was tested, which had lost expression ofHLA molecules A29, B44, and C clone 10. The subline was lysed, thusindicating that the presenting molecule should be one of Al, B37, orCw6.

Example 2

Further studies were carried out to determine if 76/6 also producedtumor necrosis factor ("TNF") when contacted with target cells. Themethod used was that described by Traversari et al., Immunogenetics 35:145-152 (1992), the disclosure of which is incorporated by reference.Briefly, samples of the CTL line were combined with samples of a targetcell of interest in culture medium. After 24 hours, supernatant from thecultures was removed, and then tested on TNF-sensitive WEHI cells. Cellline MZ2MEL.43, a subclone of the MZ2-MEL cell line discussed supra aswell as in the cited references, gave an extremely strong response, andwas used in the following experiments.

Example 3

The results from Example 2 indicated that MZ2.MEL.43 presented thetarget antigen of interest. As such, it was used as a source of totalmRNA to prepare a CDNA library.

Total RNA was isolated from the cell line. The mRNA was isolated usingan oligo-dT binding kit, following well recognized techniques. Once theMRNA was secured, it was transcribed into cDNA, via reversetranscription, using an oligo dT primer containing a NotI site, followedby second strand synthesis. The CDNA was then ligated to a BstXIadaptor, digested with NotI, size fractionated on a Sephacryl S-500 HRcolumn, and then cloned, undirectionally, into the BstXI and Not I sitesof pcDNAI/Amp. The recombinant plasmid was then electroporated into DH5αE. coli bacteria. A total of 1500 pools of 100 recombinant bacteria wereseeded in microwells. Each contained about 100 cDNAs, because nearly allbacteria contained an insert.

Each pool was amplified to saturation and plasmid DNA was extracted byalkaline lysis and potassium acetate precipitation, without phenolextraction.

Example 4

Following preparation of the library described in Example 3, the cDNAwas transfected into eukaryotic cells. The transfections, describedherein, were carried out in duplicate. Samples of COS-7 cells wereseeded, at 15,000 cells/well into tissue culture flat bottom microwells,in Dulbecco's modified Eagles Medium ("DMEM") supplemented with 10%fetal calf serum. The cells were incubated overnight at 37° C., mediumwas removed and then replaced by 50 μl/well of DMEM medium containing10% Nu serum, 400 μg/ml DEAE-dextran, and 100 μM chloroquine, plus 100ng of the plasmids. As was indicated supra, the lysis studies did notestablish which HLA molecule presented the antigen. As a result, cDNAfor each of the HLA molecules which could present the antigen (A1, B37,Cw6) was used, separately, to cotransfect the cells. Specifically, oneof 28 ng of the gene encoding HLA-A1, cloned into pCD-SRα 50 ng of cDNAfor HLA-B37 in pcDNA-I-Amp, or 75 ng of cDNA for HLA-Cw6 in pcDNA-I-Amp,using the same protocol as was used for transfection with the library,were used.

Transfection was carried out in duplicate wells, but only 500 pools ofthe HLA-Cw6 transfectants could be tested in single wells. Followingfour hours of incubation at 37° C., the medium was removed, and replacedby 50 μl of PBS containing 10% DMSO. This medium was removed after twominutes and replaced by 200 μl of DMEM supplemented with 10% FCS.

Following this change in medium, COS cells were incubated for 24-48hours at 37° C. Medium was then discarded, and 1000-3000 cells of CTLclone 76/6 were added, in 100 μl of Iscove's medium containing 10%pooled human serum supplemented with 20-30 U/ml of IL-2. Supernatant wasremoved after 24 hours, and TNF content was determined in an assay onWEHI cells, as described by Traversari et al., Immunogenetics 35:145-152 (1992), the disclosure of which is incorporated by reference.

The 1500 pools transfected with HLA-A1, and the 1500 pools transfectedwith HLA-B37 stimulated TNF release to a concentration of 15-20 pg/ml,or 2-6 pg/ml, respectively. Most of the HLA-Cw6 transfectants yielded3-20 pg/ml, except for one pool, which yielded more than 60 pg/ml. Thispool was selected for further work.

Example 5

The bacteria of the selected pool were cloned, and 600 clones weretested. Plasmid DNA was extracted therefrom, transfected into a newsample of COS cells in the same manner as described supra, and the cellswere again tested for stimulation of CTL clone 76/6.Ninety-four-positive clones were found. One of these, referred to ascDNA clone 2D6, was tested further. In a comparative test COS cells weretransfected with cDNA clone 2D6 and the HLA-Cw6 cDNA, HLA-Cw6 cDNAalone, or cDNA 2D6 alone. Control cell lines MZ2-MEL F⁻ and MZ2-MEL F⁺were also used. TNF release into CTL supernatant was measured by testingit on WEHI cells, as referred to supra. The number of surviving WEHIcells was measured by optical density after incubation of the cells withMTT. FIG. 2 shows that the COS cells transfected with HLA-Cw6 andcDNA-2D6, and the cell line MZ2-MEL F⁺ stimulated TNF release from CTLclone 76/6, indicating that HLA-Cw6 presented the subject TRA.

Example 6

The CDNA 2D6 was sequenced following art known techniques. A sequencesearch revealed that the plasmid insert showed no homology to knowngenes or proteins. SEQ ID NO: 1 presents CDNA nucleotide information forthe identified gene, referred to hereafter as "GAGE". A putative openreading frame is located at bases 51-467 of the molecule. The first twobases of this sequence are from the vector carrying the CDNA sequence,and are thus not part of the cDNA itself.

Example 7

Following sequencing of the cDNA, as per Example 6, experiments-werecarried out to determine if cells of normal tissues expressed the gene.To determine this, Northern blotting was carried out on tissues andtumor cell lines, as indicated below. The blotting experiments used cDNAfor the complete sequence of SEQ ID NO: 1. PCR was then used to confirmthe results.

                  TABLE 1                                                         ______________________________________                                        Expression of gene GAGE                                                       ______________________________________                                        Normal tissues                                                                                     PHA activated T cells -                                    CTL clone 82/30 -                                                             Liver -                                                                       Muscle -                                                                      Lung -                                                                        Brain -                                                                       Kidney -                                                                      Placenta -                                                                    Heart -                                                                       Skin - Testis +                                                               Tumor cell lines                                                              Melanoma  7/16                                                                Lung Carcinoma 1/6                                                            Sarcoma 0/1                                                                   Thyroid medullary carcinoma 0/1                                               Tumor samples                                                                 Melanoma 1/1                                                                ______________________________________                                    

Example 8

Detailed analysis of normal tissues and tumors was carried out byapplying polymerase chain reaction ("PCR") and the GAGE gene informationdescribed supra.

First, total RNA was taken from the particular sample, using artrecognized techniques. This was used to prepare cDNA. The protocol usedto make the cDNA involved combining 4 ul of reverse transcriptase buffer5×, 1 ul of each dNTP, (10 mM), 2 ul of dithiothreitol (100 mM), 2 ul ofdT-15 primer (20 um), 0.5 ul of RNasin (40 units/ul), and 1 ul of MOMLVreverse transcriptase (200 units/ul). Next, 6.5 ul of template RNA (1ug/3.25 ul water, or 2 ug total template RNA) was added. The totalvolume of the mixture was 20 ul. This was mixed and incubated at 42° C.for 60 minutes, after which it was chilled on ice. A total of 80 ul ofwater was then added, to 100 ul total. This mixture was stored at -20°C. until used in PCR.

To carry out PCR, the primers

    5'-AGA CGC TAC GTA GAG CCT-3'

(sense)

and

    5'-CCA TCA GGA CCA TCT TCA-3'

(antisense).

SEQ ID NOS: 2 and 3, respectively, were used. The reagents included 30.5ul water, 5 ul of PCR buffer 10×, 1 ul of each dNTP (10 uM), 2.5 ul ofeach primer (20 uM), and 0.5 ul of polymerizing enzyme Dynazyme (2units/ul). The total volume was 45 ul. A total of 5 ul of cDNA was added(this corresponded to 100 ng total RNA). The mixture was combined, andlayered with one drop of mineral oil. The mixture was transferred to athermocycler block, preheated to 94° C., and amplification was carriedout for 30 cycles, each cycle consisting of the following:

    ______________________________________                                        first denaturation: 94° C., 4 min.                                       denaturation: 94° C., 1 min.                                           annealing: 55° C., 2 min.                                              extension: 72° C., 3 min.                                              final extension: 72° C., 15 min.                                     ______________________________________                                    

Following the cycling, 10 ul aliquots were run on a 1.5% agarose gel,stained with ethidium bromide.

CDNA amplified using the primers set forth supra yields a 238 base pairfragment. There is no amplification of contaminating genomic DNA, ifpresent.

The results are presented in Table 2, which follows. They confirm thatthe only normal tissue which expresses GAGE is testis, whereas a numberof tumors, including melanoma, lung, breast, larynx, pharynx, sarcoma,testicular seminoma, bladder and colon express the gene. Thus, any oneof these tumors can be assayed for by assaying for expression of theGAGE gene.

                  TABLE 2                                                         ______________________________________                                        RT-PCR analysis of the expression of gene GAGE                                ______________________________________                                               NORMAL TISSUES                                                           Heart -                                                                       Brain -                                                                       Liver -                                                                       Lung -                                                                        Kidney -                                                                      Spleen -                                                                      Lymphocytes -                                                                 Bone marrow -                                                                 Skin -                                                                        Naevus -                                                                      Melanocytes -                                                                 Fibroblasts -                                                                 Prostate -                                                                    Testis +                                                                      Ovary -                                                                       Breast -                                                                      Adrenals -                                                                    Muscle -                                                                      Placenta -                                                                    Umbilical Cord -                                                            ______________________________________                                        TUMORS         Cell lines Tumor samples                                       ______________________________________                                        Melanoma       40/63      46/146  (32%)                                         Lung cancer                                                                   Epidermoid carcinoma  10/41   (24%)                                           Adenocarcinoma  4/18                                                          Small Cell Lung Cancer  6/23 0/2                                              Breast cancer  15/146 (10%)                                                   Head and Neck tumor                                                           Larynx  6/15 (40%)                                                            Pharynx  3/13                                                                 Sarcoma 1/4 6/18 (33%)                                                        Testicular seminoma  6/6  (100%)                                              Bladder cancer  2/20                                                          Prostate cancer  2/20                                                         Colon carcinoma  5/13 0/38                                                    Renal cancer 0/6 0/45                                                         Leukemia 3/6 0/19                                                           ______________________________________                                    

Example 9

The identification of the nucleic acid molecule referred to in the priorexamples led to further work directed to the determination of tumorrejection antigens presented by HLA-Cw6 molecules, and derived from theGAGE gene.

The complete CDNA of GAGE in expression vector pcDNAI/Amp was digestedwith restriction endonucleases NotI and SpHI, and then with exonucleaseIII following supplier's instruction (Erase-a-base System, Promega).This treatment generated a series of progressive deletions, starting atthe 3'end.

The deletion products were ligated back into pcDNAI/Amp, and thenelectroporated into E. coli strain DH5alpha'IQ, using well knowntechniques. The transformants were selected with ampicillin (50micrograms/ml).

Plasmid DNA was extracted from each recombinant clone and was thentransfected into COS-7 cells, together with a vector which coded forHLA-Cw6. The protocols used follow the protocols described above.

The transfectants were then tested in the TNF release assay. Thispermitted separation of positive and negative clones. All the negativeclones showed a deletion of the entire GAGE sequence. The smallestpositive clone contained the first 170 nucleotides of SEQ ID NO: 1. Theanalysis of this sequence, supra, notes that the open reading framestarts at nucleotide 51. Thus, this fragment contains a sequence whichencodes the first 40 amino acids of the GAGE TRAP.

Example 10

Additional experiments were then carried out to define the regionencoding the TRA peptide more precisely. Polymerase chain reaction("PCR") amplification was used to do this.

Two primers were synthesized. The first primer was a 22-mercomplementary to a sequence within the plasmid vector pcDNAI/Amp locatedupstream of a BamHI site. The second primer was a 29-mer containing atthe 3'end nucleotides 102-119 of SEQ ID NO: 1, and at the 5'end anextension of 11 nucleotides containing an XbaI restriction site.

Following amplification, the PCR product was digested by BamHI and XbaI,and cloned into the BamHI-XbaI sites of plasmid pcDNA-3. The recombinantcolonies were cotransfected into COS-7 cells with cDNA encoding HLA-Cw6,in accordance with Example 4, and a TNF release assay, also as describedsupra, was carried out, using CTL 76/6.

TNF release was observed, indicating that the "minigene" was processedto a TRA. The minigene, i.e., nucleotides 1-119 of SEQ ID NO: 1, thecoding region of which runs from nucleotides 51-119 encoded the first 23amino acids of the cDNA of SEQ ID NO: 1. This information served as thebasis for the next set of experiments.

Example 11

Two peptides were synthesized, based upon the first 23 amino acids ofSEQ ID NO: 1. These were:

    Met Ser Trp Arg Gly Arg Ser Thr Tyr Arg Pro Arg Pro Arg Arg (SEQ ID NO: 12)

and

    Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val Glu Pro Pro Glu Met Ile (SEQ ID NO: 13)

Each peptide was pulsed into COS-7 cells previously transfected withHLA-Cw6 cDNA, and combined with CTL 76/6 to determine if TNF releasewould be induced. Peptides (20 ug/ml) were added to COS-7 cells whichhad been transfected with the HLA-Cw6 cDNA twenty-four hours previously.After incubation at 37° C. for 90 minutes, medium was discarded, and3000 CTLs were added in 100 microliters of medium, containing 25units/ml of IL-2. Eighteen hours later, TNF content of supernatant wastested via determining toxicity on WEHI-164-13 cells. The second peptide(SEQ ID NO: 13) was found to induce more than 30 pg/ml of TNF, while thefirst peptide (SEQ ID NO: 12), was found to induce less than 10 pg/ml ofTNF. The second peptide was used for further experiments.

Example 12

Various peptides based upon SEQ ID NO: 13 were synthesized, and tested,some of which are presented below. To carry out these tests, ⁵¹ Crlabelled LB33-EBV cells, which are HLA-Cw6 positive, were incubated withone of the following peptides:

    Tyr Arg Pro Arg Pro Arg Arg Tyr                                                                       (SEQ ID NO: 4)                                           - Thr Tyr Arg Pro Arg Pro Arg Arg Tyr (SEQ ID NO: 5)                          - Tyr Arg Pro Arg Pro Arg Arg Tyr Val (SEQ ID NO: 6)                          - Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val (SEQ ID NO: 7)                      - Arg Pro Arg Pro Arg Arg Tyr Val Glu (SEQ ID NO: 8)                          - Met Ser Trp Arg Gly Arg Ser Thr Tyr Arg (SEQ ID NO:12)                      - Pro Arg Pro Arg Arg                                                  

The peptide concentration varied, as indicated in FIG. 3, and the ratioof CTL: LB33-EBV ("effector: target ratio"), was 10:1. ⁵ Cr release wasdetermined after four hours of incubation at 37° C. Levels of lysis forpositive ("F⁺ ", MZ2-MEL.3.1), and negative ("F⁻ "; MZ2-MEL.2.2.5)control cells are indicated, in FIG. 3.

It was found, quite surprisingly, that the octamer of SEQ ID NO: 4 wasthe best peptide, and appeared to be the tumor rejection antigen. Thisis the first time an octamer has been reported as being involved inpresentation by a human MHC molecule. There is some precedent for amurine system, as reported by Engelhard, supra, at 199, for H-2K^(b) andH-2K^(K) molecules. The nonamers of SEQ ID NO: 5 and SEQ ID NO: 6 alsoinduced CTL lysis albeit to a lesser extent than the octamer of SEQ IDNO: 4.

In results not reported here, a second CTL was tested (CTL 82/31). ThisCTL was known to lyse cells presenting MZ2-F. It, too, lysed HLA-Cw6positive cells following pulsing with the peptide of SEQ ID NO: 4.

Example 13

To find out whether the GAGE DNA set forth supra was unique, a CDNAlibrary made with RNA from MZ2-MEL.43 (the same library that was usedfor the cloning of GAGE) was hybridized with a probe derived from theGAGE CDNA. The probe was a PCR fragment of 308 base pairs betweenpositions 20 and 328 of SEQ ID NO: 1. Twenty positive cDNAs wereobtained. Six of them were entirely sequenced. They were all highlyrelated to the GAGE sequence, but they were slightly different from it.Two of the six clones were identical to each other, but all the othersdiffered from each other. Thus, five new sequences different from buthighly related to GAGE were identified. They are called GAGE-2, 3, 4, 5and 6 (FIG. 4), and are presented as SEQ ID NOS: 14-18, respectively.The fourteen other clones were partially sequenced at the 5' end andtheir sequence corresponded to one of the six GAGE cDNAs.

The major difference between these cDNAs and GAGE-1 is the absence of astretch of 143 bases located at position 379 to 521 of the GAGE sequenceof SEQ ID NO: 1. The rest of the sequences shows mismatches only at 19different positions, with the exception of GAGE-3 whose 5'end is totallydifferent from the other GAGE for the first 112 bases. This region ofthe GAGE-3 cDNA contains a long repeat and a hairpin structure.

The deduced GAGE-1 protein corresponding to a tumor rejection antigenprecursor is about 20 amino acids longer than the 5 other proteins,whose last seven residues also differ from the homologous residues ofGAGE-1 (FIG. 5). The rest of the protein sequences show only 10mismatches. One of these is in the region corresponding to the antigenicpeptide of SEQ ID NO: 4. The sequence of the peptide is modified inGAGE-3, 4, 5 and 6 so that position 2 is now W instead of R.

Example 14

To assess whether the change at position 2 affected the antigenicity ofthe peptide, cDNA of the 6 GAGE cDNAs were individually transfected intoCOS cells together with the CDNA of HLA-Cw6, and the transfectants weretested for recognition by CTL 76/6 as described, supra. Only GAGE-1 andGAGE-2 transfected cells were recognized, showing that the modifiedpeptide encoded by GAGE-3, 4, 5 and 6 was not antigenic in the contextof this experiment. Sequence analysis of the 5' end of the 14 otherclones mentioned supra, showed that 7 of them contained the sequenceencoding the antigenic peptide, and thus probably corresponded to eitherGAGE-1 or GAGE-2.

Example 15

The PCR primers used, supra to test the expression of GAGE in tumorsamples do not discriminate between GAGE-1 or 2 and the four other GAGEcDNAs that do not encode antigen MZ2F. A new set of primers was preparedwhich specifically amplifies GAGE-1 and 2, and not GAGE-3, 4, 5 and 6.These primers are:

    VDE44 5'-GAC CAA GAC GCT ACG TAG-3' (SEQ ID NO: 9)

    VDE24 5'-CCA TCA GGA CCA TCT TCA-3' (SEQ ID NO: 10)

These primers were used as described, supra, in a RT-PCR reaction usinga polymerase enzyme in the following temperature conditions:

    ______________________________________                                         4 min at 94° C.                                                         30 cycles with 1 min at 94                                                                 ° C.                                                       2 min at 56° C.                                                        3 min at 72° C.                                                       15 min at 72° C.                                                     ______________________________________                                    

The results of this analysis are set forth in Table 3.

                  TABLE 3                                                         ______________________________________                                        Expression of GAGE genes by tumor samples and tumor cell lines                              Number of GAGE positive tumors                                  Histological type                                                                           All GAGE genes*                                                                             GAGE-1 and 2**                                    ______________________________________                                        Tumor samples                                                                   Melanomas                                                                     primary lesions 5/39 5/39 (13%)                                               metastases 47/132 36/131 (27%)                                                Sarcomas 6/20   6/20 (30%)                                                    Lung carcinomas NSCLC 14/65  12/64 (19%)                                      Head and neck squamous 13/55  10/54 (19%)                                     cell carcinomas                                                               Prostatic carcinomas 2/20 2/20                                                Mammary carcinomas 18/162 14/162  (9%)                                        Bladder carcinomas                                                            superficial 1/20 1/20                                                         infiltrating 5/26 3/26                                                        Testicular seminomas 6/6  5/6                                                 Colorectal carcinomas 0/43                                                    Leukemias and lymphomas 0/25                                                  Renal carcinomas 0/46                                                         Tumor cell lines                                                              Melanomas 45/74  40/74 (54%)                                                  Sarcomas 1/4  1/4                                                             Lung carcinomas                                                               SCLC 7/24 7/24 (29%)                                                          NSCLC 1/2  1/2                                                                Mesotheliomas 5/19 5/19 (26%)                                                 Head and neck squamous 0/2                                                    cell carcinomas                                                               Mammary carcinomas 1/4  0/4                                                   Bladder carcinomas 0/3                                                        Colon carcinomas 5/13 5/13                                                    Leukemias 3/6  1/6                                                            Lymphomas 0/6                                                                 Renal carcinomas 0/6                                                         *Expression of GAGE was tested by RTPCR on total RNA with primers VDE18       and VDE24, detecting all GAGE genes. No PCR product was observed when         these primers were assayed on DNA from MZ2MEL.                                **Expression of GAGE1 and 2 was tested by RTPCR on total RNA with primers     VDE44 and VDE24, which distinguish GAGE1 and 2 from the four other GAGE       genes. No PCR product was observed when these primers were assayed on DNA     from MZ2MEL.                                                             

In further work, new primers were designed which amplified all GAGEgenes, to make sure that there was no expression of any of them innormal tissues. These primers are

    VDE43 5'-GCG GCC CGA GCA GTT CA-3' (SEQ ID NO: 11)

    VDE24 5'-CCA TCA GGA CCA TCT TCA-3 (SEQ ID NO: 10)

These were used exactly as for the PCR using the VDE44 and VDE24primers. The results are shown in Table 4. They confirm that the normaltissues are negative, except for testis.

                  TABLE 4                                                         ______________________________________                                        Expression of GAGE genes in normal adult and fetal tissues                                   GAGE                                                             expression*                                                                 ______________________________________                                        Adult tissues                                                                   Adrenal gland -                                                               Benign naevus -                                                               Bone marrow -                                                                 Brain -                                                                       Breast -                                                                      Cerebellium -                                                                 Colon -                                                                       Heart -                                                                       Kidney -                                                                      Liver -                                                                       Lung -                                                                        Melanocytes -                                                                 Muscle -                                                                      Ovary -                                                                       Prostate -                                                                    Skin -                                                                        Splenocytes -                                                                 Stomach -                                                                     Testis +                                                                      Thymocytes -                                                                  Urinal bladder -                                                              Uterus -                                                                      Placenta -                                                                    Umbilical -                                                                   cord                                                                          Fetal tissues.sup.                                              Fibroblasts -                                                                 Brain -                                                                       Liver -                                                                       Spleen -                                                                      Thymus -                                                                      Testis +                                                                    ______________________________________                                         *Expression of GAGE was tested by RTPCR amplification on total RNA with       primers VDE43 and VDE24 detecting all GAGE genes (FIG. 7). Absence of PCR     product is indicated by - and presence by +. No PCR product was observed      when these primers were assayed on DNA from MZ2MEL.                           .sup. Fetal tissues derive from fetuses older than 20 weeks

The foregoing examples show the isolation of nucleic acid moleculeswhich code for tumor rejection antigen precursors and tumor rejectionantigens. These molecules, however, are not homologous with any of thepreviously disclosed MAGE and BAGE coding sequences described in thereferences set forth supra. Hence, one aspect of the invention is anisolated nucleic acid molecule which comprises the nucleotide sequenceset forth in SEQ ID NO: 1 as well as fragments thereof, such asnucleotides 1-170, and 51-170, and any other fragment which is processedto a tumor rejection antigen. The sequence of SEQ ID NO: 1 is neither aMAGE nor a BAGE coding sequence, as will be seen by comparing it to thesequence of any of these genes as described in the cited references.Also a part of the invention are those nucleic acid molecules which alsocode for a non-MAGE and non-BAGE tumor rejection antigen precursor butwhich hybridize to a nucleic acid molecule containing the describednucleotide sequence, under stringent conditions. The term "stringentconditions" as used herein refers to parameters with which the art isfamiliar. More specifically, stringent conditions, as used herein,refers to hybridization in 1M NaCl, 1% SDS, and 10% dextran sulfate.This is followed by two washes of the filter at room temperature for 5minutes, in 2× SSC, and one wash for 30 minutes in 2× SSC, 0.1% SDS.There are other conditions, reagents, and so forth which can be used,which result in the same or higher degree of stringency. The skilledartisan will be familiar with such conditions, and, thus, they are notgiven here.

It will also be seen from the examples that the invention embraces theuse of the sequences in expression vectors, as well as to transform ortransfect host cells and cell lines, be these prokaryotic (e.g., E.coli), or eukaryotic (e.g., CHO or COS cells). The expression vectorsrequire that the pertinent sequence, i.e., those described supra, beoperably linked to a promoter. As it has been found that human leukocyteantigen HLA-Cw6 presents a tumor rejection antigen derived from thesegenes, the expression vector may also include a nucleic acid moleculecoding for HLA-Cw6. In a situation where the vector contains both codingsequences, it can be used to transfect a cell which does not normallyexpress either one. The tumor rejection antigen precursor codingsequence may be used alone, when, e.g., the host cell already expressesHLA-Cw6. Of course, there is no limit on the particular host cell whichcan be used. As the vectors which contain the two coding sequences maybe used in HLA-Cw6 presenting cells if desired, and the gene for tumorrejection antigen precursor can be used in host cells which do notexpress HLA-Cw6.

The invention also embraces so called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

To distinguish the nucleic acid molecules and the TRAPs of the inventionfrom the previously described MAGE and BAGE materials, the inventionshall be referred to as the GAGE family of genes and TRAPs. Hence,whenever "GAGE" is used herein, it refers to the tumor rejection antigenprecursors coded for by the previously described sequences. "GAGE codingmolecule" and similar terms, are used to describe the nucleic acidmolecules themselves.

The invention as described herein has a number of uses, some of whichare described herein. First, the invention permits the artisan todiagnose a disorder such as melanoma, characterized by expression of theTRAP, or presentation of the tumor rejection antigen. These methodsinvolve determining expression of the TRAP gene, and/or TRAs derivedtherefrom, such as a TRA presented by HLA-Cw6. In the former situation,such determinations can be carried out via any standard nucleic aciddetermination assay, including the polymerase chain reaction, orassaying with labelled hybridization probes. In the latter situation,assaying with binding partners for complexes of TRA and HLA, such asantibodies, is especially preferred. An alternate method fordetermination is a TNF release assay, of the type described supra. Tocarry out the assay, it is preferred to make sure that testis cells arenot present, as these normally express GAGE. This is not essential,however, as one can routinely differentiate between testis and othercell types. Also, it is practically impossible to have testis cellspresent in non-testicular sample.

The isolation of the TRAP gene also makes it possible to isolate theTRAP molecule itself, especially TRAP molecules containing the aminoacid sequence coded for by SEQ ID NO: 1. These isolated molecules whenpresented as the TRA, or as complexes of TRA and HLA, such as HLA-Cw6,may be combined with materials such as adjuvants to produce vaccinesuseful in treating disorders characterized by expression of the TRAPmolecule. In addition, vaccines can be prepared from cells which presentthe TRA/HLA complexes on their surface, such as non-proliferative cancercells, non-proliferative transfectants, et cetera. In all cases wherecells are used as a vaccine, these can be cells transfected with codingsequences for one or both of the components necessary to provide a CTLresponse, or be cells which express both molecules without transfection.Further, the TRAP molecule, its associated TRAs, as well as complexes ofTRA and HLA, may be used to produce antibodies, using standardtechniques well known to the art.

When "disorder" is used herein, it refers to any pathological conditionwhere the tumor rejection antigen precursor is expressed. An example ofsuch a disorder is cancer, melanoma in particular. Melanoma is wellknown as a cancer of pigment producing cells.

As indicated, supra, tumor rejection antigens, such as the one presentedin SEQ ID NO: 4 are also a part of the invention. Also a part of theinvention are polypeptides, such as molecules containing from 8 to 16amino acids, where the polypeptides contain the amino acid sequence setforth in SEQ ID NO: 4. As the examples indicate, those peptides whichare longer than the octamer of SEQ ID NO: 4 are processed into the tumorrejection antigen of SEQ ID NO: 4 by the HLA-Cw6 presenting cancercells, and presented thereby. The presentation leads to lysis bycytolytic T lymphocytes present in a body fluid sample contacted to thecells presenting the complex. The fact that these peptides are processedto the tumor rejection antigen, is indicated by the examples.

This property may be exploited in the context of other parameters inconfirming diagnosis of pathological conditions, such as cancer,melanoma in particular. For example, the investigator may study antigensshed into blood or urine, observe physiological changes, and thenconfirm a diagnosis of melanoma using the CTL proliferationmethodologies described herein.

On their own, peptides in accordance with the invention may be used tocarry out HLA-typing assays. It is well known that when a skin graft,organ transplant, etc., is necessary one must perform HLA typing so asto minimize the possibility of graft rejection. The peptides of theinvention may be used to determine whether or not an individual isHLA-Cw6 positive, so that appropriate donors may be selected. This typeof assay is simple to carry out. The peptides of the invention arecontacted to a sample of interest, and binding to cells in that sampleindicates whether or not the individual from which the sample is takenis HLA-Cw6 positive. One may label the peptides themselves, conjugate orotherwise bind them to linkers which are labeled, immobilize them tosolid phases, and so forth, so as to optimize such an assay. Otherstandard methodologies will be clear to the skilled artisan, and neednot be presented herein.

Therapeutic approaches based upon the disclosure are premised on aresponse by a subject's immune system, leading to lysis of TRApresenting cells, such as HLA-Cw6 cells. One such approach is theadministration of CTLs specific to the complex to a subject withabnormal cells of the phenotype at issue. It is within the skill of theartisan to develop such CTLs in vitro. Specifically, a sample of cells,such as blood cells, are contacted to a cell presenting the complex andcapable of provoking a specific CTL to proliferate. The target cell canbe a transfectant, such as a COS cell of the type described supra. Thesetransfectants present the desired complex on their surface and, whencombined with a CTL of interest, stimulate its proliferation. COS cells,such as those used herein are widely available, as are other suitablehost cells.

To detail the therapeutic methodology, referred to as adoptive transfer(Greenberg, J. Immunol. 136(5): 1917 (1986); Riddel et al., Science 257:238 (Jul. 10, 1992); Lynch et al., Eur. J. Immunol. 21: 1403-1410(1991); Kast et al., Cell 59: 603-614 (Nov. 17, 1989)), cells presentingthe desired complex are combined with CTLs leading to proliferation ofthe CTLs specific thereto. The proliferated CTLs are then administeredto a subject with a cellular abnormality which is characterized bycertain of the abnormal cells presenting the particular complex, wherethe complex contains the pertinent HLA molecule. The CTLs then lyse theabnormal cells, thereby achieving the desired therapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA/TRA complex. This can bedetermined very easily, as the art is very familiar with methods foridentifying cells which present a particular HLA molecule, as well ashow to identify cells expressing RNA of the pertinent sequences, in thiscase a GAGE sequence. Once cells presenting the relevant complex areidentified via the foregoing screening methodology, they can be combinedwith a sample from a patient, where the sample contains CTLs. If thecomplex presenting cells are lysed by the mixed CTL sample, then it canbe assumed that a GAGE derived, tumor rejection antigen is beingpresented, and the subject is an appropriate candidate for thetherapeutic approaches set forth supra.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo, usinga number of approaches. One approach, i.e., the use of non-proliferativecells expressing the complex, has been elaborated upon supra. The cellsused in this approach may be those that normally express the complex,such as irradiated melanoma cells or cells transfected with one or bothof the genes necessary for presentation of the complex. Chen et al.,Proc. Natl. Acad. Sci. USA 88: 110-114 (January, 1991) exemplifies thisapproach, showing the use of transfected cells expressing HPV E7peptides in a therapeutic regime. Various cell types may be used.Similarly, vectors carrying one or both of the genes of interest may beused. Viral or bacterial vectors are especially preferred. In thesesystems, the gene of interest is carried by, e.g., a Vaccinia virus orthe bacteria BCG, and the materials de facto "infect" host cells. Thecells which result present the complex of interest, and are recognizedby autologous CTLs, which then proliferate. A similar effect can beachieved by combining the tumor rejection antigen or the precursoritself with an adjuvant to facilitate incorporation into HLA-Cw6presenting cells which then present the HLA/peptide complex of interest.The TRAP is processed to yield the peptide partner of the HLA moleculewhile the TRA is presented without the need for further processing.

Other aspects of the invention will be clear to the skilled artisan andneed not be repeated here.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  18                                         - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 646 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CTGCCGTCCG GACTCTTTTT CCTCTACTGA GATTCATCTG TGTGAAATAT  - #                  50                                                                        - - GAGTTGGCGA GGAAGATCGA CCTATCGGCC TAGACCAAGA CGCTACGTAG  - #                 100                                                                         - - AGCCTCCTGA AATGATTGGG CCTATGCGGC CCGAGCAGTT CAGTGATGAA  - #                 150                                                                         - - GTGGAACCAG CAACACCTGA AGAAGGGGAA CCAGCAACTC AACGTCAGGA  - #                 200                                                                         - - TCCTGCAGCT GCTCAGGAGG GAGAGGATGA GGGAGCATCT GCAGGTCAAG  - #                 250                                                                         - - GGCCGAAGCC TGAAGCTGAT AGCCAGGAAC AGGGTCACCC ACAGACTGGG  - #                 300                                                                         - - TGTGAGTGTG AAGATGGTCC TGATGGGCAG GAGATGGACC CGCCAAATCC  - #                 350                                                                         - - AGAGGAGGTG AAAACGCCTG AAGAAGAGAT GAGGTCTCAC TATGTTGCCC  - #                 400                                                                         - - AGACTGGGAT TCTCTGGCTT TTAATGAACA ATTGCTTCTT AAATCTTTCC  - #                 450                                                                         - - CCACGGAAAC CTTGAGTGAC TGAAATATCA AATGGCGAGA GACCGTTTAG  - #                 500                                                                         - - TTCCTATCAT CTGTGGCATG TGAAGGGCAA TCACAGTGTT AAAAGAAGAC  - #                 550                                                                         - - ATGCTGAAAT GTTGCAGGCT GCTCCTATGT TGGAAAATTC TTCATTGAAG  - #                 600                                                                         - - TTCTCCCAAT AAAGCTTTAC AGCCTTCTGC AAAGAAAAAA AAAAAA   - #                    646                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - AGACGCTACG TAGAGCCT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - CCATCAGGAC CATCTTCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE:  amino aci - #d                                                     (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - Tyr Arg Pro Arg Pro Arg Arg Tyr                                                           5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE:  amino aci - #d                                                     (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - Thr Tyr Arg Pro Arg Pro Arg Arg Tyr                                                       5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE:  amino aci - #d                                                     (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - Tyr Arg Pro Arg Pro Arg Arg Tyr Val                                                       5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE:  amino aci - #d                                                     (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val                                                   5 - #                  - #10                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE:  amino aci - #d                                                     (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                           - - Arg Pro Arg Pro Arg Arg Tyr Val Glu                                                       5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - GACCAAGACG CTACGTAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - CCATCAGGAC CATCTTCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - GCGGCCCGAG CAGTTCA             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 12:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE:  amino aci - #d                                                     (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                          - - Met Ser Trp Arg Gly Arg Ser Thr Tyr Arg Pr - #o Arg Pro Arg Arg                           5 - #                  - #10                  - #15           - -  - - (2) INFORMATION FOR SEQ ID NO: 13:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE:  amino aci - #d                                                     (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                          - - Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val Gl - #u Pro Pro Glu Met Ile                       5 - #                  - #10                  - #15           - -  - - (2) INFORMATION FOR SEQ ID NO: 14:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 538 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14:                          - - ACGCCAGGGA GCTGTGAGGC AGTGCTGTGT GGTTCCTGCC GTCCGGACTC  - #                  50                                                                         - - TTTTTCCTCT ACTGAGATTC ATCTGTGTGA AATATGAGTT GGCGAGGAAG  - #                 100                                                                         - - ATCGACCTAT CGGCCTAGAC CAAGACGCTA CGTAGAGCCT CCTGAAATGA  - #                 150                                                                         - - TTGGGCCTAT GCGGCCCGAG CAGTTCAGTG ATGAAGTGGA ACCAGCAACA  - #                 200                                                                         - - CCTGAAGAAG GGGAACCAGC AACTCAACGT CAGGATCCTG CAGCTGCTCA  - #                 250                                                                         - - GGAGGGAGAG GATGAGGGAG CATCTGCAGG TCAAGGGCCG AAGCCTGAAG  - #                 300                                                                         - - CTCATAGCCA GGAACAGGGT CACCCACAGA CTGGGTGTGA GTGTGAAGAT  - #                 350                                                                         - - GGTCCTGATG GGCAGGAGAT GGACCCGCCA AATCCAGAGG AGGTGAAAAC  - #                 400                                                                         - - GCCTGAAGAA GGTGAAAAGC AATCACAGTG TTAAAAGAAG ACACGTTGAA  - #                 450                                                                         - - ATGATGCAGG CTGCTCCTAT GTTGGAAATT TGTTCATTAA AATTCTCCCA  - #                 500                                                                         - - ATAAAGCTTT ACAGCCTTCT GCAAAGAAAA AAAAAAAA      - #                      - #    538                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 15:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 560 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #15:                          - - CTCATATTTC ACACAGATGA GTTGGCGAGG AAGATCGACC TATTATTGGT  - #                  50                                                                         - - CTAGGCCAAT AATAGGTCGA TCTTCCTCGC CAACTCATAT TTCACACAGA  - #                 100                                                                         - - TGAATCTCAG TAGAGGAAAA TCGACCTATT ATTGGCCTAG ACCAAGGCGC  - #                 150                                                                         - - TATGTACAGC CTCCTGAAGT GATTGGGCCT ATGCGGCCCG AGCAGTTCAG  - #                 200                                                                         - - TGATGAAGTG GAACCAGCAA CACCTGAAGA AGGGGAACCA GCAACTCAAC  - #                 250                                                                         - - GTCAGGATCC TGCAGCTGCT CAGGAGGGAG AGGATGAGGG AGCATCTGCA  - #                 300                                                                         - - GGTCAAGGGC CGAAGCCTGA AGCTGATAGC CAGGAACAGG GTCACCCACA  - #                 350                                                                         - - GACTGGGTGT GAGTGTGAAG ATGGTCCTGA TGGGCAGGAG ATGGACCCGC  - #                 400                                                                         - - CAAATCCAGA GGAGGTGAAA ACGCCTGAAG AAGGTGAAAA GCAATCACAG  - #                 450                                                                         - - TGTTAAAAGA AGGCACGTTG AAATGATGCA GGCTGCTCCT ATGTTGGAAA  - #                 500                                                                         - - TTTGTTCATT AAAATTCTCC CAATAAAGCT TTACAGCCTT CTGCAAAGAA  - #                 550                                                                         - - AAAAAAAAAA                - #                  - #                      - #       560                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 16:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 540 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #16:                          - - CGCCAGGGAG CTGTGAGGCA GTGCTGTGTG GTTCCTGCCG TCCGGACTCT  - #                  50                                                                         - - TTTTCCTCTA CTGAGATTCA TCTGTGTGAA ATATGAGTTG GCGAGGAAGA  - #                 100                                                                         - - TCGACCTATT ATTGGCCTAG ACCAAGGCGC TATGTACAGC CTCCTGAAAT  - #                 150                                                                         - - GATTGGGCCT ATGCGGCCCG AGCAGTTCAG TGATGAAGTG GAACCAGCAA  - #                 200                                                                         - - CACCTGAAGA AGGGGAACCA GCAACTCAAC GTCAGGATCC TGCAGCTGCT  - #                 250                                                                         - - CAGGAGGGAG AGGATGAGGG AGCATCTGCA GGTCAAGGGC CGAAGCCTGA  - #                 300                                                                         - - AGCTGATAGC CAGGAACAGG GTCACCCACA GACTGGGTGT GAGTGTGAAG  - #                 350                                                                         - - ATGGTCCTGA TGGGCAGGAG ATGGACCCGC CAAATCCAGA GGAGGTGAAA  - #                 400                                                                         - - ACGCCTGAAG AAGGTGAAAA GCAATCACAG TGTTAAAAGA AGGCACGTTG  - #                 450                                                                         - - AAATGATGCA GGCTGCTCCT ATGTTGGAAA TTTGTTCATT AAAATTCTCC  - #                 500                                                                         - - CAATAAAGCT TTACAGCCTT CTGCAAAAAA AAAAAAAAAA     - #                      - #   540                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 17:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 532 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #17:                          - - AGCTGTGAGG CAGTGCTGTG TGGTTCCTGC CGTCCGGACT CTTTTTCCTC  - #                  50                                                                         - - TACTGAGATT CATCTGTGTG AAATATGAGT TGGCGAGGAA GATCGACCTA  - #                 100                                                                         - - TTATTGGCCT AGACCAAGGC GCTATGTACA GCCTCCTGAA GTGATTGGGC  - #                 150                                                                         - - CTATGCGGCC CGAGCAGTTC AGTGATGAAG TGGAACCAGC AACACCTGAA  - #                 200                                                                         - - GAAGGGGAAC CAGCAACTCA ACGTCAGGAT CCTGCAGCTG CTCAGGAGGG  - #                 250                                                                         - - AGAGGATGAG GGAGCATCTG CAGGTCAAGG GCCGAAGCCT GAAGCTGATA  - #                 300                                                                         - - GCCAGGAACA GGGTCACCCA CAGACTGGGT GTGAGTGTGA AGATGGTCCT  - #                 350                                                                         - - GATGGGCAGG AGATGGACCC GCCAAATCCA GAGGAGGTGA AAACGCCTGA  - #                 400                                                                         - - AGAAGGTGAA AAGCAATCAC AGTGTTAAAA GAAGGCACGT TGAAATGATG  - #                 450                                                                         - - CAGGCTGCTC CTATGTTGGA AATTTGTTCA TTAAAATTCT CCCAATAAAG  - #                 500                                                                         - - CTTTACAGCC TTCTGCAAAG AAAAAAAAAA AA       - #                  - #             532                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 18:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 539 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #18:                          - - GCCAGGGAGC TGTGAGGCAG TGCTGTGTGG TTCCTGCCGT CCGGACTCTT  - #                  50                                                                         - - TTTCCTCTAC TGAGATTCAT CTGTGTGAAA TATGAGTTGG CGAGGAAGAT  - #                 100                                                                         - - CGACCTATTA TTGGCCTAGA CCAAGGCGCT ATGTACAGCC TCCTGAAGTG  - #                 150                                                                         - - ATTGGGCCTA TGCGGCCCGA GCAGTTCAGT GATGAAGTGG AACCAGCAAC  - #                 200                                                                         - - ACCTGAAGAA GGGGAACCAG CAACTCAACG TCAGGATCCT GCAGCTGCTC  - #                 250                                                                         - - AGGAGGGAGA GGATGAGGGA GCATCTGCAG GTCAAGGGCC GAAGCCTGAA  - #                 300                                                                         - - GCTGATAGCC AGGAACAGGG TCACCCACAG ACTGGGTGTG AGTGTGAAGA  - #                 350                                                                         - - TGGTCCTGAT GGGCAGGAGG TGGACCCGCC AAATCCAGAG GAGGTGAAAA  - #                 400                                                                         - - CGCCTGAAGA AGGTGAAAAG CAATCACAGT GTTAAAAGAA GACACGTTGA  - #                 450                                                                         - - AATGATGCAG GCTGCTCCTA TGTTGGAAAT TTGTTCATTA AAATTCTCCC  - #                 500                                                                         - - AATAAAGCTT TACAGCCTTC TGCAAAAAAA AAAAAAAAA      - #                      - #   539                                                                    __________________________________________________________________________

We claim:
 1. Isolated nucleic acid molecule consisting of nucleotides1-170 of SEQ ID NO:
 1. 2. Isolated nucleic acid molecule consisting ofnucleotides 1-119 of SEQ ID NO:
 1. 3. Expression vector comprising theisolated nucleic acid molecule of claim 1, operably linked to apromoter.
 4. Cell line comprising the isolated nucleic acid molecule ofclaim
 1. 5. Cell line comprising the expression vector of claim
 3. 6.Isolated nucleic acid molecule consisting of nucleotides 51-170 of SEQID NO:
 1. 7. Expression vector comprising the isolated nucleic acidmolecule of claim 6, operably linked to a promoter.
 8. Cell linecomprising the isolated nucleic acid molecule of claim
 6. 9. Cell linecomprising the expression vector of claim
 7. 10. The cell line of claim4, 5, 8 or 9, wherein said cell line expresses an HLA-Cw6 molecule. 11.The cell line of claim 9 wherein said cell line has been transfectedwith a nucleic acid molecule which codes for HLA-Cw6.
 12. Biologicallypure prokaryotic cell transformed with the nucleic acid molecule ofclaim
 6. 13. Biologically pure prokaryotic cell transformed with theexpression vector of claim 7.